Julita Ramírez

Opposing effects of SWI/SNF and Mi-2/NuRD chromatin remodeling complexes on epigenetic reprogramming by EBF and Pax5

Transcriptionally silent genes are maintained in inaccessible chromatin. Accessibility of these genes requires their modification by chromatin remodeling complexes (CRCs), which are recruited to promoters by sequence-specific DNA-binding proteins. Early B-cell factor (EBF), which is crucial for B-cell lineage specification, reprograms mb-1 (Ig-α) promoters by increasing chromatin accessibility and initiating the loss of DNA methylation. In turn, this facilitates promoter activation by Pax5. Here, we investigated the roles of ATP-dependent CRCs in these mechanisms. Fusion of EBF and Pax5 with the ligand-binding domain of ERα allowed for 4-hydroxytamoxifen-dependent, synergistic activation of mb-1 transcription in plasmacytoma cells. Knock-down of the SWI/SNF ATPases Brg1 and Brm inhibited transcriptional activation by EBF:ER and Pax5:ER. In contrast, knock-down of the Mi-2/NuRD complex subunit Mi-2β greatly enhanced chromatin accessibility and mb-1 transcription in response to the activators. The reduction of Mi-2β also propagated DNA demethylation in response to EBF:ER and Pax5:ER, resulting in fully unmethylated mb-1 promoters. In EBF- or EBF/Pax5-deficient fetal liver cells, both EBF and Pax5 were required for efficient demethylation of mb-1 promoters. Together, our data suggest that Mi-2/NuRD is important for the maintenance of hypermethylated chromatin in B cells. We conclude that SWI/SNF and Mi-2/NuRD function in opposition to enable or limit the reprogramming of genes by EBF and Pax5 during B-cell development.

From hematopoietic progenitors to B cells: mechanisms of lineage restriction and commitment

The generation of B lymphocytes from hematopoietic progenitors requires lineage-specific transcription factors that progressively direct cell fate choices. Differentiation of hematopoietic stem cells to lymphoid progenitors requires Ikaros-dependent lineage priming and graded levels of PU.1, which are controlled by Ikaros and Gfi1. E2A drives expression of EBF1, which initiates B lineage specification. EBF1, in addition to Pax5, is necessary for commitment to the B cell lineage. As a model of gene activation in early B lymphopoiesis, mb-1 genes are activated sequentially by factors (e.g. EBF1) that initiate chromatin modifications before transcription. This review highlights the requisite interplay between transcription factors and epigenetic mechanisms in the context of B cell development.

Bivalent recognition of nucleosomes by the tandem PHD fingers of the CHD4 ATPase is required for CHD4-mediated repression

CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis for their association with chromatin. We found that PHD1/2 targets nucleosomes in a multivalent manner, concomitantly engaging two histone H3 tails. This robust synergistic interaction displaces HP1γ from pericentric sites, inducing changes in chromatin structure and leading to the dispersion of the heterochromatic mark H3K9me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.

The Mi-2/NuRD complex: a critical epigenetic regulator of hematopoietic development, differentiation and cancer.

The Mi-2/NuRD chromatin remodeling complex links multiple transcriptional regulatory processes including histone deacetylation, histone demethylation, nucleosome mobilization and recruitment of other regulatory proteins. In some contexts, Mi-2/NuRD functions as a barrier to transcriptional activation by working in opposition to other chromatin remodelers such as SWI/SNF. Alternatively, the Mi-2β ATPase subunit of Mi-2/NuRD can promote transcription. Together, these gatekeeper functions of Mi-2/NuRD influence cell fate decisions by modulating transcriptional activity. Recent studies have shown the importance of Mi-2/NuRD both in maintaining hematopoietic stem cell (HSC) pools and in normal lineage progression. Furthermore, components of Mi-2/NuRD complexes have been linked directly to oncogenesis and have potential as therapeutic targets for cancer treatment. Mi-2/NuRD’s essential functions in metazoan cell fates and activities underscore its importance as a focal point of epigenetic research.

Compound haploinsufficiencies of Ebf1 and Runx1 genes impede B cell lineage progression

Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation of Cd79a (mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences of Ebf1 haploinsufficiency together with haploinsufficiency of Runx1 genes in mice. Although numbers of “committed” pro-B cells were maintained in Ebf1+/−Runx1+/− (ERhet) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, κ0 sterile transcripts, CD2 and CD25 were reduced and delayed in ERhet pro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells in ERhet mice. Late pre-B and immature and mature B cells were decreased in the bone marrow of Ebf1+/− (Ehet) mice and were nearly absent in ERhet mice. Although we did not observe significant effects of haploinsuficiencies on IgH or Igκ rearrangements, a relative lack of Igλ rearrangements was detected in Ehet and ERhet pre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow of Ehet and ERhet mice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reduced Ebf1 dosage and the compounding effects of reduced Runx1 dosage. Our data confirm and extend the importance of EBF1 in regulating target genes and Ig gene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.

MBD2 and multiple domains of CHD4 are required for transcriptional repression by Mi-2/NuRD complexes

ABSTRACT Mi-2/nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complexes are important regulators of chromatin structure and DNA accessibility. We examined requirements for individual domains of chromodomain helicase DNA-binding protein 4 (CHD4), a core catalytic component of NuRD complexes, as well as the NuRD subunit methyl-binding domain protein 2 (MBD2) and methylated DNA, for NuRD function in the context of tissue-specific transcription. By itself, loss of NuRD activity is not sufficient for transcriptional activation. However, NuRD complexes greatly reduce activation of the B cell-specific mb-1 (Cd79a) gene by the transcription factors EBF1 and Pax5. Using our B cell model system, we determined that the two chromodomains and ATPase/helicase and C-terminal domains (CTD) of CHD4 are all necessary for repression of mb-1 promoters by NuRD. All of these domains except the CTD are required for efficient association of CHD4 with mb-1 promoter chromatin. Loss of MBD2 expression or of DNA methylation impaired association of CHD4 with mb-1 promoter chromatin and enhanced its transcription. We conclude that repressive functions of MBD2-containing NuRD complexes are dependent on cooperative interactions between the major domains of CHD4 with histones and DNA and on binding of methylated DNA by MBD2.

The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis

BACKGROUND Cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone. Cyp11a1 expression is increased in activated T cells. OBJECTIVES We sought to determine the role of Cyp11a1 activation in the development of peanut allergy and TH cell functional differentiation. METHODS A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on T(H)1, T(H)2, and T(H)17 differentiation were determined. Cyp11a1 gene silencing was performed with Cyp11a1-targeted short hairpin RNA. RESULTS Peanut sensitization and challenge resulted in diarrhea, inflammation, and increased levels of Cyp11a1, IL13, and IL17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG treatment decreased IL13 and IL17A mRNA expression in the small intestine without affecting Cyp11a1 mRNA or protein levels. In vitro the inhibitor decreased IL13 and IL17A mRNA and protein levels in differentiated T(H)2 and T(H)17 CD4 T cells, respectively, without affecting GATA3, retinoic acid-related orphan receptor γt (RORγt), or T(H)1 cells and IFNG and T-bet expression. Short hairpin RNA-mediated silencing of Cyp11a1 in polarized T(H)2 CD4 T cells significantly decreased pregnenolone and IL13 mRNA and protein levels. CONCLUSION Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T(H)2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.